1. Field of the Invention
The invention relates to a .gamma.-phosphate linked adenosine 5' triphosphate (ATP)-Sepharose affinity column for the purification of protein kinases and a method for using the affinity column to purify protein kinases.
2. Background of the Invention
Protein kinases are enzymes capable of phosphorylating certain amino acid residues in specific proteins. For example, the breakdown of glycogen is regulated by protein kinases. Specifically, when adrenaline binds to the liver cell, cyclic adenosine monophosphate (AMP) is produced which promotes the activation of cyclic AMP dependent protein kinase. Phosphorylation of the active form of glycogen synthase (dephospho) by cyclic AMP protein kinase converts the enzyme to its phosphorylated, inactive form. The transformation of glycogen synthase to its phosphorylated form allows for the breakdown of glycogen to glucose to be used for energy. Cyclic AMP dependent protein kinase further catalyzes the phosphorylation of the inactive phosphorylase kinase by ATP to yield the active, phosphorylated form of phosphorylase kinase.
Protein kinases are allosteric enzymes. Cyclic AMP dependent protein kinases represent a typical example of this group of enzymes. The inactive forms contain two catalytic subunits and two regulatory subunits. When the subunits are associated together in a complex, the enzyme is inactive. However, when cyclic AMP binds to the sites of the two regulatory subunits, the protein kinase becomes enzymatically active. When the protein kinase is activated, the enzyme can phosphorylate a number of other enzymes in different kinds of target cells.
Therefore, the process of protein phosphorylation is recognized as a fundamental mechanism by which regulation of many important cellular processes is achieved.
Extensive research has been devoted to the characterization of protein kinases which catalyze protein phosphorylation, due to the linkage of many of these enzymes with diseases such as cancer and diabetes. The enzymes may also be of agricultural and industrial importance since several protein kinases have been identified which regulate plant, yeast and bacterial metabolism in, for example, rapeseed oil synthesis, brewing, and antibiotic synthesis.
Protein kinases can be categorized into three distinct classes, based on the amino acids they phosphorylate. These are the serine/threonine protein kinases, eg. cyclic AMP-dependent protein kinase (Walsh et al. (1968) J. Biol. Chem., 243:3763-3765; Taylor et al. (1990) in Peptides and Protein Phosphorylation, (Kemp, B. E. ed) pp. 2-32, CRC Press) or mitogen-activated protein (MAP) kinase (Sturgill et al. (1991) Biochem. Biophys. Acta., 1092:350-357); the tyrosine protein kinases, eg. p60.sup.c-arc (Anderson et al. (1985) Mol. Cell. Biol., 5:1122-1128; Martinez et al. (1987) Science, 237: 411-415); and the recently discovered dual specificity protein kinases that phosphorylate exogenous substrates on both tyrosine and serine/threonine amino acids, eg. MAP kinase kinase (MAPKK) (Ahn et al. (1991) J. Biol. Chem., 266:4220-4227; Gomez et al. (1991) Nature, 353:170-175; Crews et al. (1992) Science, 258: 478-480; Seger et al. (1992) J. Biol. Chem., 267:25688-25631; Seger et al. (1992) J. Biol. Chem., 267:14373-14381; Nakielny et al. (1992) FEBS Lett., 308:183-189; Ashworth et al. (1992) Oncogene, 7:2555-2556; Wu et al. (1992) Biochem, J. 285: 701-705.
However, despite the growing interest in protein kinases, to date, no direct methods have existed for the affinity purification of protein kinases.
Past attempts to purify protein kinases have resulted in relatively poor purification steps. ATP columns such as ATP-agarose have been used in many protein kinase purification protocols.
However, typically these columns have been constructed by coupling the nucleotide to a solid matrix, either through the N6 amino group on the purine ring or hydroxyl groups of the ribose moiety {Trayer et al. (1974) Biochem. J., 139:609-623; Jeno, P. and Thomas, G (1991) Methods Enzymology Zoo: 178-187. However, based on the orientation of MgATP within the catalytic cleft of cyclic A kinase, previous ATP columns were unlikely to act as true affinity steps in the purification of most, if not all, protein kinases. The reason is that the purine rings are buried in a hydrophobic pocket, sterically precluding affinity purification by linkage through these moieties. Linkage via the ribose hydroxyl groups is also likely to be sterically hindered from viewing the structure of cyclic A kinase.
Thus, previous ATP columns have functioned for the most part as weak cation-exchange resins and not as a specific affinity ligand for protein kinases.
Further, although purification of up to twenty-fold has been obtained in a few cases (e.g., 70-kDa ribosomal S6 kinase described in Jeno et al. (1989) J. Biol. Chem., 264:1293-1297; Jeno et al. (1991) Methods Enzymol., 200:178-187), with such columns, adsorption requires low ionic strength, suggesting binding of protein kinases by ionic interactions only.
Therefore, a more effective means of immobilizing ATP is needed in order to provide a means for purification of protein kinases.